Field of the Technology
The disclosure relates to the field of resistive pulse sensing, specifically to a process and method for analyzing the size, charge, shape, mechanical and other properties of micro- and nano-sized particles, including biological cells and viruses.
Description of the Prior Art
Resistive-pulse sensing involves the detection and analysis of particles as they pass through a channel or pore separating two reservoirs of electrolyte solution. The resistance of that pore is monitored by applying a voltage between the reservoirs, which drives a flux of ions through it, detected as a current flowing from the voltage source. Particles may be driven through the pore electrokinetically thus by electroosmosis and/or electrophoresis, by pressure, or simply move by diffusion, and they modulate the flux of ions as they pass through, thus inducing pulses in the measured resistance. In addition to counting particles, analysis of these pulses allows estimation of particle size, electrophoreticmobility, effective charge, and measurement of the size and volume of the pore used to analyze those particles.
Single-pore technology has been used to detect particles and determine their size. The approach works well for objects of very different sizes. Single nanopores with diameters less than 10 nm are used to detect single molecules such as DNA and proteins. Pores with openings of several tens and several hundreds of nanometers were shown to detect viral capsids and particles. Pores of micrometer size are routinely used in complete blood counts at hospitals. In all the above-mentioned cases, the species to be detected were in contact with one side of a single-pore membrane that separated two chambers of a conductivity cell filled with an electrolyte. Single molecules, particles, and cells were detected during their passage through the pore as a transient change in the recorded ion current called the resistive pulse.
Passing of particles through pores is also very important in the context of drug delivery, when considering particle clearance from the body. Hydrogels have become the center of interest since it was shown that by applying a pressure difference these deformable particles could pass through pores whose diameters were much smaller than the effective particle size. If used as drug-delivery vehicles, these particles could therefore be cleared through the kidney system, which is known to contain nanopores with effective opening diameters of approximately 8 nm. Clearing particles through the renal system is preferred since it prevents particle accumulation in the liver, which would otherwise lead to hepatoxicity. Transport experiments of 116 nm diameter hydrogels were performed with polymer membranes containing multiple pores with an average opening of 10 nm.
Detecting deformable particles with single pores could potentially provide more information than measurements with many-pore membranes. By studying resistive pulses one could learn about the dynamics of the particle deformation on a single-particle basis. Since multi-pore membranes contain pores with a finite distribution of the pore diameters, having one pore of known geometry allows one to understand the relation between the pore opening diameter and the pressure required for the particle deformation. Studies with single man-made pores and hydrogels have been performed under an applied pressure difference using glass pipettes, with openings between 200 and 700 nm, and 570 nm diameter hydrogel particles. Transport of single hydrogels led to the formation of a unique pattern of the resistive pulses consisting of a current increase followed by a current drop below the baseline value. Since the particles were largely filled with salt solution and additionally carried surface charges, their approach to the pore opening resulted in the increase of the measured transmembrane current. In order to squeeze through an opening that was smaller than the particle size, the particles had to deform and dehydrate, which was observed as a current decrease. The pulse shape was found to be dependent on the value of the applied pressure difference and the pore diameter. If the particles passed through sufficiently wide pipettes, the pulse consisted of only one positive peak, indicating that the presence of the particle in the pore lowered the system resistance. It is important to mention that in this system deformation of particles could be observed only in cases when the particles were passing through pipettes with openings smaller than the particles' diameter.
The use of single nanopores in detecting particles and biological cells is known and has been documented. See for example U.S. Pat. No. 2,656,508, DeBlois, R. W., Bean, C. P.; Wesley, R. K. A. “Electrokinetic Measurements with Submicron Particles and Pores by the Resistive Pulse Technique,”J. Colloid Interface Sci. 1977, 61, 323-335; and DeBlois, R. W.; Bean, C. P. “Counting and Sizing of Submicron Particles by the Resistive Pulse Technique,” Rev. Sci. Instrum. 1970, 41, 909-916.
The first example of resistive-pulse sensing was the Coulter counter developed to count and size blood cells. The Coulter counter has since been used to characterize a variety of analytes, including bacteria, mitochondria, viruses and gas bubbles. With the advent of track-etched pores, resistive pulse sensing was extended to counting and sizing nanoscale particles, such as polystyrene spheres and viruses. Later, ion channels enabled sensing of polymers and small molecules as well as of nucleic acids and proteins and are now on the cusp of sequencing DNA. Recently, resistive-pulse sensing has been demonstrated with solid-state nanopores, silica nanochannels, gold nanoconstriction, and PDMS nanochannels. The central part of the Coulter counter device comprises a single pore which gets transiently occluded when single particles (e.g. biological cells) pass through under the influence of an applied pressure difference and electric field.
The commercial unit of the Beckman Coulter counter is applicable for the detection and counting of blood cells. The detection is based on the difference in volume of various cells. The technique used by the commercial Beckman Coulter is thus not capable of detecting cells of different shapes. The commercially available unit does not characterize mechanical properties of any transport objects and cannot perform any affinity studies, e.g. distinguishing cells based on their ability to bind to specific agents. Flow cytometry is a more versatile device allowing studies of cells and particles' chemical affinity, size and even sub-cellular structures but often requires staining of the cells, which compromises their viability. There is currently no tool on the market capable of high-throughput characterization of mechanical properties of cells and particles. None of the tools offer a possibility of simultaneous characterization of size, shape, mechanical properties and chemical affinities on a single object level.
In addition, the commercial unit cannot be used to detect circulating tumor cells (CTCs), which are shed from a tumor site into the blood stream. CTCs are extremely rare (a few or a few tens of cells per 10 mL of blood) and thus cannot be detected within the large background of the blood cells. Moreover, the Beckman Coulter counter requires even further dilution of the blood samples. There has been a lot of interest in detecting and analyzing CTCs since their presence is related with malignancy of tumors and their response to therapy. CTCs also give insight into the heterogeneity of cancer cells. If a sufficient number of CTCs were isolated, various anticancer drugs could be tested to design personal and more efficient treatment for each patient.
It was noted in the past that electrical fluctuations within a resistive pulse correspond to physical variations in the structure of the pore. However, this has seen little application, used only to determine the base and tip diameters of a conical pore and to reveal the tapered shape of glass nanopipettes. In addition, although surfactant is routinely added to the particle solution to prevent aggregation and mitigate pore clogging, the significant impact of surfactant on particle velocity has never been reported.
Microfluidic channels have been successfully applied to detect CTCs. Some of the existing microfluidic devices are based on the volume of CTCs which is often larger compared to the volume of red and white blood cells; other devices combine the size and deformability of CTCs, for example as disclosed by W. Zhang, K. Kai, D. S. Choi, T. Iwamoto, Y. H. Nguyen, H. Wong, M. D. Landis, N. T. Ueno, J. Chang, L. Qin, Proc. Natl. Acad. Sci. U.S.A. 2012, 109(46), 18707-18712; S. C. Hur, N. K. Henderson-MacLennan, E. R. B. McCabe, D. Di Carlo, Lab Chip 2011, 11(5), 912-920; review: I. Cima, C. W. Yee, F. S. Iliescu, W. M. Phyo, K. H., Lim, C. Iliescu, M. H. Tan. Biomicrofluidics 2013, 7, 011810 (1-17)]. Another microfluidic system to characterize CTCs by their size and mass as well as deformability has been reported by Byun et al. in Proc. Natl. Acad. Sci. USA doi: 10.1073/pnas.1218806110 (2013). These approaches require the cells to pass through constrictions significantly smaller than the cells, which can compromise the cells' viability.
The only FDA approved system for detecting CTCs (CellSearch, Veridex, LLC) uses the presence of Epithelial Cell Adhesion Molecule (EpCAM) on the cell surface and the binding of cells to an antibody towards EpCAM. However, since not all CTCs over express EpCAM, this method is not capable of detecting all CTCs and cells originating from different primary tumors.
The current resistive pulse technique used in the art is performed from diluted solutions, which slows down the analysis. In addition, prior art methods cannot distinguish between particles of different shapes, but only similar volume and charge. Detecting shape is relevant for the detection of viruses and cells, since misshapen cells can be indicative of disease. Current resistive-pulse techniques also cannot characterize cells by their mechanical properties, and cannot be applied for the detection of circulating tumor cells (CTC).
What is needed is a device and method that involves using single pores with varying cross-sectional sizes and pore roughness that leads to repeatable signatures of ion current being recorded when the particles are translocating the pore. A platform is needed which is capable of increasing the throughput of the Coulter counter approach by at least an order of magnitude with less or no dilution.
The needed platform should be able to simultaneously characterize each individual cell with multiple physical properties including size, shape, surface charge and deformability. Characterization of the physical properties together with characterization of chemical affinities is important as well.